1. Field of the Invention
The present invention relates to polymers used with light producing stabilized 1,2-dioxetanes or other chemiluminescent compounds to produce enhanced chemiluminescence. More particularly, the present invention relates to polymeric ammonium and phosphonium salts with or without fluorescent molecules and methods of manufacture therefor. Even more particularly, the present invention concerns polymeric phosphonium and ammonium salts prepared from the reaction of (a) polyvinylbenzyl chloride having (1) added π-electrons or (2) higher molecular weight and/or (3) both and (b) trisubstituted amines and/or trisubstituted phosphines. The present invention, also concerns describes the use of these new polymers to enhance the chemiluminescent light produced by the decomposition of stabilized 1,2-dioxetanes which can be destabilized by the action of an enzyme or a chemical.
2. Prior Art
As is known to those skilled in the art to which the present invention pertains, enhancers are substances which increase the amount of chemiluminescence emitted by a 1,2-dioxetane or other chemiluminescent system. The best known chemiluminescent reactions are those which employ, stabilized, enzyme-triggerable 1,2-dioxetanes, acridanes, acridinium esters, luminol, isoluminol and derivatives thereof or lucigenin. These chemiluminescent compounds are generally denoted in the art as either agents, reactants or substrates. Enhanced chemiluminescence means that the total light emitted, the maximum light intensity and/or the ratio of light intensity of the reaction compared to the background is greater than that observed in the absence of the enhancer.
The effect of a non-chemiluminescent donor on luminescence has been found to be profoundly influenced by changes in the ratio between the luminescent donor and the non-luminescent donor. For example, the increase of light emission caused by the addition of o-phenylenediamine to a peroxidase-based system is accompanied by an accelerated formation of the blue dimer, thus speeding up the rate-limiting step. Further, at constant pyrogallol concentrations, it has been found that the addition of o-phenylenediamine never causes an increase in the integrated luminescence yield, although the velocity of the luminescent process can be accelerated, thus giving a higher luminescence peak. On the other hand incorporation of p-phenylenediamine into a peroxidase-purpurogallin-hydrogen peroxide system results in an eight-fold enhancement of light emission [M. Halmann, B. Velan, T. Sery and H. Schupper, Photochem. Photobiochem., 30, 165 (1979)]. The effect of other factors such as pH change, heavy water, radical scavengers and the addition of other enhancers on the enhancement of chemiluminescence of peroxidase-catalyzed reactions has been reported in the literature. See, inter alia, L. Ewetz and A. Thore, Anal. Biochem., 71, 564 (1976); J. K. Wong and M. L. Salin, Photochem. Photobiol., 33, 737 (1981); H. P. Misra and P. M. Squatrioto, Arch. Biochem. Biophys., 215, 59 (1982); G. H. G. Thorpe, L. J. Kricka, E. Gillespie, S. Moseley, R. Amess, N. Bagget and T. P. Whitehead, Anal. Biochem., 145, 96 (1985); T. J. N. Carter, C. J. Groucutt, R. A. W. Stott, G. H. G. Thorpe and T. P. Whitehead, European Patent, No. 87959 (1982); G. H. G. Thorpe, L. J. Kricka, S. B. Moseley and T. P. Whitehead, Clin. Chem. (Winston-Salem, N.C.), 31, 1335 (1985); L. J. Kricka, G. H. G. Thorpe and T. P. Whitehead, European Patent No. 116454 (1983) and U.S. Pat. No. 4,598,044.
Similarly, numerous assay enhancers have been employed in conjunction with and in a peroxidase-catalyzed reaction of luminol or acridane to increase the intensity and duration of light emission. These enhancers include benzothiazole derivatives such as 6-hydroxybenzothiazole derivatives, dehydroluciferin, firefly luciferin, substituted phenols such as p-iodophenols, p-phenylphenol or 2-naphthol, and aromatic amines such as p-phenylenediamine or tetramethyl benzidine. Other compounds which function as enhancers for chemiluminescent oxidation of amino-substituted cyclic acylhydrazide by a peroxidase include N,N-dimethylindoaniline, 2,6-dichlorophenoline-o-cresol, phenolindophenol, N-methyl-phenathiazine and a combination of phenolindolphenol and N-methylphenathiazine, as disclosed in U.S. Pat. No. 5,171,668 and in PCT/US97/06422 (1999), the disclosures of which are hereby incorporated by reference.
The chemiluminescence enhancing effect of 2-hydroxy-9-fluoro-4-hydroxy-3-[3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-2H-1-benzopyrene-2-one and substituted oxazole derivatives in a peroxidase-oxidant-luminol or isoluminol system has, also, been reported. See, U.S. Pat. No. 5,206,149. Similarly, surfactants including nonionic, cationic and anionic as well as polymeric compounds are known to affect the light producing efficiency of peroxidase-catalyzed reactions, as reported by L. J. Kricka and M. Deluca, Arch. Biochem. Biophys., 217, 674 (1983); T. Goto and H. Fukatsu, Tet. Letts., 4299 (1969); and K. Sasamoto and Y. Ohkura, Chem. Pharm. Bull., 39, 411 1991). The major advantage of enhanced assays is that the intensity of light emission may be greater than 1000-fold that of an un-enhanced reaction. Also, conditions can be employed under which light emission is prolonged and decay is slow.
The full mechanism for the oxidation of cyclic acylhydrazides and acridanes by the combination of peroxide and peroxidase enzyme and the light enhancement by enhancers is not known. However, many compounds reported to increase light emission from chemiluminescent and bioluminescent systems do not enhance the peroxidase-catalyzed system under reported conditions, but 6-hydroxybenzothiazole and phenol derivatives produce dramatic increases in light intensity, thereby suggesting they operate by a different mechanism. See, for example, T. P. Whitehead, G. H. G. Thorpe, T. J. N. Carter, C. Groucutt and L. J. Kricka, Nature (London), 305, 158 (1983); H. W. Yurow and S. Sass, Anal. Chim. Acta., 88, 389 (1977); D. E. Bause and H. H. Patterson, Anal. Chem., 51, 2288 (1985); F. Kohen, J. B. Kim, G. Barnard and H. R. Linder, Steroids, 36, 405 (1980); H. R. Schroeder, P. O. Vogelhut, R. J. Carrico, R. C. Boguslaski and R. T. Buckler, Anal. Chem., 48, 1933 (1976), and M. L. Grayeski and E. Woolf, in “Analytical Application of Bioluminescence and Chemiluminescence” (L. J. Kricka, P. E. Stanley, G. H. G. Thorpe and T. P. Whitehead, eds), p. 565, Academic Press, Orlando, 1984.
Similarly, numerous enhancers have been employed in conjunction with the use of stabilized 1,2-dioxtanes. The enhancement of chemiluminescence from a stable 1,2-dioxetane triggered decomposition by an enzyme in the presence of water-soluble substances including a long chain aliphatic ammonium surfactant and a fluorescent compound has been taught in U.S. Pat. No. 4,959,182. According to this reference, micelles containing cetyltrimethyl-ammonium bromide (CTAB) and a fluorescent molecule attached to a long chain hydrocarbon, 5-(N-tetradecanoyl) aminofluorescein, capture the intermediate hydroxy-substituted 1,2-dioxetane, which is destabilized under basic pH of buffer to decompose, and leads to a 400-fold increase in chemiluminescence efficiency. Enhancement occurs by virtue of an efficient intermolecular energy transfer process from the anionic form of the excited state ester to the fluorescent compound, which is held in close proximity, and the hydrophobic environment created by the surfactant.
The synthesis of polymeric quaternary ammonium compounds and polymeric benzyltrialkylphosphonium salts is well known in the literature, see, inter alia, U.S. Pat. Nos. 2,780,604; 3,178,396; 3,770,439; 3,898,088; 4,308,335; 4,340,522; 4,424,326; 4,563,411; and 3,239,519. Recently, these polymers have been used as an enhancer to enhance the light output of stabilized 1,2-dioxetanes when triggered to destabilize either chemically or enzymatically. See U.S. patent application Ser. No. 09/883,586, the disclosure of which is hereby incorporated by reference.
The effect of substitution on the solubility of certain polyvinybenzyl chloride polymers has been described in U.S. Pat. No. 4,308,335. According to this reference, the quaternary nitrogen polymers of the reference are prepared by the reaction of polyvinylbenzyl chloride and a tertiary amine. The structure of the polymer can be shown as below:
Where R1, R2, R3, R4 and R5 are, individually, either hydrogen or alkyl having from 1 to 20 carbon atoms. When the quaternary nitrogen of formula (1) has a long alkyl chain i.e. more than a five carbon chain, the resulting cationic polymer is insoluble in water but is soluble in organic solvents.
In the same way when polyvinylbenzyl chloride is treated with trialkylphosphine, the following polymer is obtained:
When the quaternary nitrogen of formula (2) has a long alkyl chain, i.e. more than five carbon atoms, the resulting cationic polymer is water-insoluble. Other water-insoluble or partially water-insoluble polymers can be synthesized by the combination of two or three different trialkylphosphines. These polymers have shown excellent enhancement of chemiluminescent of stabilized 1,2-dioxetanes compared to water-soluble polymers.
Water-soluble polymeric quaternary ammonium salts when used alone or when admixed with fluorescein, as disclosed in U.S. Pat. Nos. 4,978,614; 5,145,772; 5,547,836; 5,593,828 and 5,654,154 and polyvinylbenzyl-trialkylphosphonium salts used alone or admixed or covalently attached to fluorescent molecules as described in U.S. Pat. Nos. 5,393,479; 5,431,845; 5,474,725; 5,582,775, have been taught to enhance chemiluminescence efficiency of stable 1,2-dioxetanes. Structurally, these polymers can be shown as:
where A is selected from the group consisting of lower alkyl containing 1 to 20 carbon atoms, aryl, aralkyl or alkyaryl groups, n and p are each an integer of from about 3 to about 15 and Fl is a fluorescent molecule attached covalently to the phenyl ring or is admixed with the polymer molecules.
The attachment of Rose-Bengal, which has structural similarity to fluorescein, to polymeric materials, has, also, been reported in literature (J. Am. Chem. Soc., 97, 3741, 1975). This polymer is used to produce singlet oxygen when irradiated with visible light in the presence of oxygen in organic solvents and can be filtered after use. The dye covalently attached to the polymer can be excited with visible light and the excited dye transfers its energy to the molecular oxygen to produce singlet oxygen which is highly reactive to unsaturated organic molecules.
However, the prior art does not disclose the use of water-insoluble or partially water-soluble polymers or organic solvent soluble polymers of ammonium and phosphonium salts with or without fluorescent molecules as an enhancer for stabilized 1,2-dioxetanes and, other chemiluminescent substrates.
The prior art, also, does not teach cross-linked polymers, having added π-electrons or higher molecular weight or both, derived from polyvinylbenzy halide or copolymers of polyvinyl naphthalene and polyvinyl benzylhalide and bilinkers.
It is to this to which the present invention is directed as detailed below.